Our research focuses on mitosis and meiosis in higher
eukaryotes.
Particularly, we are interested in the
question of i) how kinesins
contribute to chromosome congression in mammalian cells ii) how
anaphase onset is controlled by the ubiquitin/
proteasome
systemiii) how
cytokinesis is synchronized with chromosome
segregation.

For these studies we combine molecular and cell biological approaches
with biochemistry and live-cell microscopy studies.
In addition to siRNAs,
we apply cell-permeable small molecule inhibitors to modulate the
activity
of proteins in living cells, an approach termed “chemical biology”.

The function of kinesins in chromosome congression

Kinesins are
molecular
motor proteins that convert energy
released by
ATP-hydrolysis into mechanical force. Recently,
we could show that
Kif18A, a kinesin-8 family member, plays
a key role in chromosome
congression
in
mammalian
cells.
Kif18A localizes to the plus-ends
of
kinetochore
microtubules
(see
Figure 1).
Cells depleted of Kif18A
show
a dramatic
delay
in prometaphase
characterized by severe
chromosome
congression
defects. Furthermore, immunofluorescence
studies revealed
that spindles
are significantly
longer
and
sister
kinetochores are under less
tension in
Kif18A-
depleted
cells.
Consistent with these in-vivo observations,
our
biochemical studies
demonstrated that Kif18A is a slow
plus-end
directed
kinesin that
depolymerizes microtubules
in a length-dependent
manner.
Thus,
Kif18A is a unique
kinesin that integrates both motility and
micro
tubule
depolymerizing activity. Currently we
are investigating
how
the
activity
of
Kif18A is regulated in time
and space.

In many vertebrates
meiosis halts at metaphase of meiosis II,
yielding
a
fertilizable or so
called “mature” egg. On fertilization
the egg quickly
enters
anaphase
and completes the second
meiotic division to
generate
a haploid
pronucleus that can then
fuse with the male
pronucleus to
form
a
diploid
zygote.
In their
classic study more than
three decades
ago
Masui
and
Markert
termed
the activity that induces
the metaphase II
arrest
“cytostatic
factor” (CSF).
Since then, CSF has
resisted its
biochemical
identification. Our
research
identified
XErp1
(Xenopus
Emi1-related
protein)
as
the cytostatic
factor
essential
for
metaphase II
arrest
in
mature ocytes.

Biochemical
assays
revealed that
XErp1
prevents
anaphase
onset by
directly inhibiting
the anaphase-promoting
complex/
cyclosome (APC),
a
ubiquitin-ligase complex that targets
cyclin-B1 and
securin
for
proteasome
dependent degradation
(Figure 2).
Studies addressing
the
mechanism of
XErp1
regulation,
identified
calcium/calmodulin-dependent
kinase II
(CaMKII) and Xenopus Polo-like
kinase
1 (Plx1)
as the key
components
initiating XErp1
destruction
upon
fertilization.
Specifically,
CaMKII
activated by
the transient
calcium
wave
triggered
by fertilization
serves
as
a
priming kinase
for Plx1
by
creating
a
docking
site for
the
polo-box domain of Plx1.
Upon binding,
Plx1
targets
XErp1
for
degradation by
phosphorylating
a so-called
phosphodegron
recognized
by
the ubiquitin-ligase
SCFb-TRCP (Figure 3).
Thus,
our
studies
not only
identified
XErp1
as
the sought-after
cytostatic
factor
but
also
revealed
how the
transient
calcium
wave triggers
anaphase
onset upon
fertilization.
Further
studies
will
focus on the
function of XErp1
in mitosis
in
mammalian
cells.

The coordination of cytokinesis with karyokinesis

The genetic integrity of each organism depends on the correct
co-
ordination
of
cytokinesis with karyokinesis (mitosis) in both
time and
space. While it is
clear
that microtubule and actin
associated proteins
are key to orchestrate
both
processes,
the
underlying mechanism
remains largely elusive. To
address
this
question, we applied a small
molecule screen to identify
compounds that specifically
target kinesins
involved in cytokinesis.
Two
compounds discovered
in these screens
selectively inhibit
the ATPase
activities of Mklp2 and MPP1. In vivo,
these compounds
induce binucleated
cells consistent with the reported
requirement
of Mklp2 and
MPP1 for proper
cytokinesis in mammalian
cells
(Figure 4). Specificity analyses
indicate
that
Mklp2 and MPP1
are
the relevant
binding partners of the identified
compoundsin vivo.
Currently, we are applying
these compounds in combination
with
live-cell microscopy studies to investigate
the specific
function of
Mklp2
and
MPP1 in cytokinesis. In parallel, we are
addressing the
mode of
action
of the
identified inhibitors by
biochemical analyses.